1. Field of the Invention
This invention relates to a method for the site specific covalent linkage or coupling of antibodies onto the substrate surface of a novel polymeric latex. The invention also relates to a method for preparing the novel latex substrate, and a method of using the coupled antibody in an immunoassay.
2. The Prior Art
In general, latex turbidimetric inhibition immunoassays utilize two reagents: (i) antibody and (ii) multivalent hapten conjugate, one of which is immobilized on the surface of a latex substrate and is commonly referred to as a "one-particle assay." Alternatively, both antibody and conjugate can also be immobilized on the latex, and this is referred to as a "two-particle assay". In the competitive latex immunoassay, antibody and conjugate reagents agglutinate in the absence of sample hapten, thereby increasing turbidity of the suspension.
The presence of hapten in a sample will inhibit the rate of agglutination, because it competes with the conjugate for the antibody binding sites. The greater the amount of hapten present in the sample, the slower the rate of increase in absorption. The reaction can be monitored, and the reaction rate of agglutination used to calculate the concentration from a rate vs. concentration curve.
Antibody-latex reagents prepared by non-site specific linkage require large amounts of antibody to obtain satisfactory agglutination with antigens, such as hapten conjugates or proteins. The need for large amounts of antibody can be attributed to the predominant attachment, on the order of about 95%, of antibody binding sites (Fab').sub.2 to the latex surface, leaving only about 5% of the binding sites free for agglutination. Consequently, such fixation reduces the specific activity of latex bound antibody. Moreover, such non-specific immobilization can contribute to serum interference, since the free Fc moiety is available to react with other agglutinators, such as rheumatoid factor (Rf) and complement (C1Q) in serum samples.
Ideally, the Fc moiety should engage the binding of antibody to the solid surface of the latex substrate thereby leaving F(ab).sub.2 free to react with haptens, conjugated haptens or proteins. This desirable site-specific attachment can be achieved by modifying the antibody, either chemically, by covalent linkage via aldehyde groups of periodate-oxidized antibody or SH group of Fab, or physically, by adsorption via "buried" hydrophobic Fc moiety. However, the chemically modified antibody may not be able to attach predominantly to a latex substrate by means of a covalent bond, since strong hydrophobic interactions can dominate slower chemical reactions. Furthermore, known chemical or physical modifications of antibody may not be attractive for large-scale commercial applications.
Latex immuno reagents are known to use poly(vinylbenzyl chloride) latex particles as the solid carrier for antibodies. These latex particles have a high refractive index and intrinsic reactive groups for bonding antibodies, proteins and oligopeptides. They perform well on Technicon RA Clinical Analyzer (Miles Inc.) systems where slight agitation of the latex is provided by periodic rotation of the reagent tray. However, when these latex reagents are used on other systems such as a Technicon Immuno 1 Clinical Analyzer (Miles Inc.), where the reagent tray turns very slowly, sedimentation of the latex particles causes erratic results, and a more frequent calibration protocol has to be adopted.
The reason for latex particle sedimentation is the high specific gravity of the latex particles in relation to the suspending agent or buffer medium. For example, state-of-the-art polyvinylbenzyl chloride latexes commonly used have a specific gravity on the order of about 1.20, which is significantly higher than the buffer medium, which has a specific gravity on the order of about 1.02. Therefore, there is a need to design `buoyant` latex particles whose performance will not be storage and system-dependent.
U.S. Pat. No. 5,095,097 to Hermentin discloses a method whereby monoclonal antibodies are covalently coupled to magnetic protein conjugates covalently without the use of amino groups. The coupling occurs at the hinge region of the antibody via thioether linkages.
U.S. Pat. No. 4,184,849 to Cambiaso discloses a method for quantitatively assaying a liquid containing small molecular weight antigens or antibodies by agglutination.
U.S. Pat. No. 4,210,723 to Dorman et al discloses a process for covalently bonding a protein with a reactive group having a labile hydrogen atom to a latex having surface epoxide groups. The preferred reactive group is a free amino group, but other reactive groups such as carboxyl, phenol, hydroxyl and thiol are also operable.
U.S. Pat. No. 4,401,765 and U.S. Pat. No. 4,480,042, both to Craig et al, disclose shell-core particles in which the high refractive index of the core results in high sensitivity to light scattering measurements and the shell contains functional groups to which antibodies, antigens, and antigen analogs can be covalently bonded.
U.S. Pat. No. 4,397,960 to Moussebois et al discloses a method for assaying a fluid that involves the following steps:
a) mixing a sample of the fluid with the F(ab').sub.2 fragments of an immunoglobulin which is specific to the antigen, to form a reaction mixture substantially free from the whole immunoglobulin and the F(c) fragments; PA1 b) incubating the mixture to allow reaction between the F(ab').sub.2 fragments and any antigen present; and PA1 c) determining the extent to which the reaction has occurred in the mixture and thereby the presence and/or amount of the antigen in the fluid sample.
U.S. Pat. No. 4,164,558 to von Schulthess et al discloses parameters such as adjustment of pH and ionic strength to maximize the sensitivity for determining the concentration of the agglutinator.
Cassart, et al, "Automated Particle-counting Immunoassay for Digoxin," Clin Chem., vol. 27, p. 1205 (1981), discloses a particle counting immunoassay (PACIA) for digoxin, based on an agglutination inhibition technique in which Rf is used as a secondary antibody to enhance the agglutination of digoxin-coated latex particles by digoxin antibody. This method requires pre-digestion of serum by pepsin in order to prevent serum interference. Other agglutinators such as C1Q, murine agglutinator (MAG) and rabbit rheumatoid factor (Rf) may be substituted for human Rf.
Masson, et al, Methods in Enzymology, vol. 74, pp. 106-139 (1981), disclose assays for digoxin that are based on a latex agglutination inhibition technique. The required sensitivity for digoxin was obtained by counting unagglutinated particles. However, these methods suffer from serum interference, which can be largely overcome, either by predigestion of serum proteins with pepsin, or by the use of F(ab').sub.2. Both particle counting and enzymatic pretreatment of samples restrict the use of these methods to special instruments and not to readily-available clinical analyzers, as well as most dedicated immunoassay systems on the market.
R. Vunnam et al., "Automated Particle Counting Immunoassay For Digoxin," Clin. Chem., vol 28, p 1656 (1982), discloses a two-particle PACIA for digoxin using digoxin-HSA-Lx (agglutinator) and Ab-Lx.
Ishikawa et al, J. Immunoassay, vol. 1 (3), pp. 385-398 (1980), discloses treatment of antibody IgG at pH 2.5 for 10 minutes and subsequent adjustment to pH 7.3-7.4 improves the performance of antibody-coated polystyrene beads in sandwich enzyme immunoassay. The Fc fragments of IgG molecules undergo conformational changes at pH 2.5 and do not appear to revert to their original structure, thus exposing more hydrophobic regions for adsorption. As a result, pH 2.5 treated IgG may be adsorbed onto the surface of polystyrene beads to heighten the efficiency of antigen binding, and/or the extent of adsorption of IgG.
Conradie et al, J. Immunol Methods, vol. 59, pp. 289-299 (1983), disclose pre-incubation of antibody at low pH will perturb the structure of the antibody molecules to expose additional hydrophobic regions. Presumably, these more hydrophobic molecules can bind to regions on the plastic surface normally not coated by non-perturbed molecules.